Method and solution for cryopreservation of umbilical cord blood and peripheral blood

ABSTRACT

An object of the present invention is to provide (i) a cryopreservation solution which can be used for cryopreserving umbilical cord blood, peripheral blood, and a component containing hematopoietic stem cells derived from any one of these types of blood and which has characteristics suitable for various utilization forms and (ii) use of the cryopreservation solution. In a cryopreservation method in accordance with the present invention, a cryopreservation solution containing a cryoprotectant and glucose is mixed with umbilical cord blood or peripheral blood, and a mixture thus obtained is frozen.

TECHNICAL FIELD

The present invention relates to a cryopreservation method and acryopreservation solution for cryopreserving umbilical cord blood andperipheral blood. More specifically, the present invention relates to acryopreservation method and a cryopreservation solution forcryopreserving umbilical cord blood and peripheral blood which methodand solution allow thawed umbilical cord blood or peripheral blood tohave good cell viability and also allow the thawed umbilical cord bloodor peripheral blood to be applied safely to a living body.

BACKGROUND ART

Umbilical cord blood and peripheral blood, each of which containshematopoietic stem cells, have been clinically applied as sources forhematopoietic stem cells, as well as bone marrow. From each of umbilicalcord blood and peripheral blood, a component containing hematopoieticstem cells is separated and recovered, if necessary, and then is mixedwith a cryopreservation solution and cryopreserved until being used.

Examples of the cryopreservation solution for cryopreserving thecomponent containing the hematopoietic stem cells encompass CP-1(product name) or CryoSure-DEX40 (product name) (Non-patent Literatures1 and 2). CP-1 contains hydroxyethylstarch and dimethyl sulfoxide asmain components, and CryoSure-DEX40 contains dextran and dimethylsulfoxide as main components.

CITATION LIST Non-patent Literature

[Non-Patent Literature 1]

Takanashi M, Oba A, Ogawa A, et al. Red blood cells depletion ofumbilical cord blood using an automated system—Evaluation of the AXPsystem. Japanese Journal of Transfusion and Cell Therapy 56(1): 62-67,2010.

[Non-Patent Literature 2]

Yamaguchi R, Takanashi M, Ito M, et al. Plasticizer concentration inumbilical cord blood cryopreserved with DMSO. Bone Marrow Transplant49(1): 157-8, 2014.

SUMMARY OF INVENTION Technical Problem

After the cryopreserved component containing the hematopoietic stemcells is thawed, the component is utilized, for example, in such amanner that the hematopoietic stem cells are separated and recovered soas to be used, or in such a manner that the component itself, togetherwith the cryopreservation solution, is applied to a living body. Assuch, there is a great demand that a cryopreservation solution forumbilical cord blood or peripheral blood, and a cryopreservationsolution for a component containing hematopoietic stem cells derivedfrom umbilical cord blood or peripheral blood, have characteristicssuitable for various utilization forms.

In relation to application to a living body, hydroxyethylstarch anddextran, which are described in Non-patent Literatures 1 and 2, arecomponents approved as drugs for medical use and are highly safe for aliving body when administered to a blood vessel of the living body.However, these components have been reported to have side effects suchas anaphylactic shock, and there is a demand for a safer component to beutilized.

Further, in a case where hematopoietic stem cells are separated andrecovered from a thawed component so as to be used, a cryoprotectivesolution is expected to have a composition that allows the hematopoieticstem cells to be easily recovered.

Further, in regard to CP-1, it is essential to add serum album to CP-1in a standard method of using CP-1. As such, especially for use inmedical situations, CP-1 still has room for improvement in terms of easeand speed in handling of CP-1.

The present invention is accomplished in order to solve the foregoingproblems. An object of the present invention is to provide (i) acryopreservation solution which can be used for cryopreserving umbilicalcord blood, peripheral blood, and a component containing hematopoieticstem cells derived from any one of these types of blood and which hascharacteristics suitable for various utilization forms and (ii) use ofthe cryopreservation solution.

Solution to Problem

A cryopreservation method in accordance with the present invention is acryopreservation method for cryopreserving umbilical cord blood orperipheral blood, including: a mixing step of mixing a cryopreservationsolution with umbilical cord blood or peripheral blood, thecryopreservation solution containing a cryoprotectant and glucose; and afreezing step of freezing a mixture obtained in the mixing step.

A production method in accordance with the present invention forproducing frozen umbilical cord blood or frozen peripheral blood is aproduction method for producing frozen umbilical cord blood or frozenperipheral blood, including: a mixing step of mixing a cryopreservationsolution with umbilical cord blood or peripheral blood, thecryopreservation solution containing a cryoprotectant and glucose, theumbilical cord blood or peripheral blood containing living cells; and afreezing step of freezing a mixture obtained in the mixing step.

The present invention also provides frozen umbilical cord blood orfrozen peripheral blood, produced by the production method above.

A cryopreservation solution in accordance with the present invention forcryopreserving umbilical cord blood or peripheral blood contains acryoprotectant and glucose.

A kit in accordance with the present invention is a kit forcryopreserving umbilical cord blood or peripheral blood, including: thecryopreservation solution above for cryopreserving umbilical cord bloodor peripheral blood; and a container which is cold resistant and capableof containing (i) the cryopreservation solution and (ii) umbilical cordblood or peripheral blood.

Advantageous Effects of Invention

The use of a cryopreservation method and a cryopreservation solution inaccordance with the present invention allows thawed umbilical cord bloodor peripheral blood to have good cell viability and also allow thethawed umbilical cord blood or peripheral blood to be applied safely toa living body.

DESCRIPTION OF EMBODIMENTS

As a result of diligent study for attaining the object, the inventors ofthe present invention have found that, in a case where umbilical cordblood is mixed with a solution containing a cryoprotectant and glucose,and a resultant mixture is frozen, the mixture when thawed has good cellviability. Through this finding, the inventors of the present inventionhave accomplished the present invention. The following description willdiscuss embodiments of the present invention in detail.

[Method for Cryopreserving Umbilical Cord Blood or Peripheral Blood]

A cryopreservation method in accordance with the present invention forcryopreserving umbilical cord blood or peripheral blood includes: amixing step of mixing a cryopreservation solution with umbilical cordblood or peripheral blood, the cryopreservation solution containing acryoprotectant and glucose; and a freezing step of freezing a mixtureobtained in the mixing step.

Umbilical cord blood applicable to the cryopreservation method inaccordance with the present invention is not limited to a particularorigin, and it is possible to preserve umbilical cord blood of placentalmammals in general. Examples of the placental mammals encompass:experimental animals such as mice, rats, rabbits, guinea pigs, andprimates other than humans; pets such as dogs and cats; farm animalssuch as bovines, horses, pigs, and ovines; and humans. Further,peripheral blood applicable to the cryopreservation method in accordancewith the present invention is not limited to a particular origin, andmay be, for example, any of the animals mentioned above. Thecryopreservation method in accordance with the present invention iseffectively applicable to human umbilical cord blood and peripheralblood.

Umbilical cord blood may be obtained, for example, by puncturing anumbilical vein and collecting the umbilical cord blood falling freelytherefrom. Peripheral blood may be collected, for example, directly byextracorporeal circulation. As such, an embodiment of thecryopreservation method in accordance with the present invention furtherincludes a step of collecting umbilical cord blood from an umbilicalcord or a step of collecting peripheral blood from a living body.

The cryoprotectant contained in the cryopreservation solution is notparticularly limited, provided that the cryoprotectant can constitute acryopreservation solution that is capable of sufficiently preservingumbilical cord blood or peripheral blood. Examples of the cryoprotectantcan encompass dimethyl sulfoxide (hereinafter, DMSO), glycerol,propylene glycol, 1-methyl-2-pyrolidone, and the like. Thecryoprotectant is preferably DMSO or propylene glycol, and particularlypreferably DMSO. The content of the cryoprotectant in the mixtureobtained in the mixing step is preferably 3.0 w/v % to 10.0 w/v %, morepreferably 5.0 w/v % to 8.0 w/v % or 5.0 v/v % to 8.0 v/v %, and from aviewpoint of cell recovery rate and reduction in the amount of DMSO,more preferably 5.0 v/v %.

The content of the glucose in the mixture obtained in the mixing step ispreferably 0.25 w/v % to 5.0 w/v %, more preferably 0.5 w/v % to 5.0 w/v%, further more preferably 1.0 w/v % to 2.5 w/v %, and particularlypreferably 1.5 w/v % to 2.0 w/v %.

In the cryopreservation solution, the ratio of concentration between thecryoprotectant and the glucose is preferably 20:1 to 1:1, morepreferably 10:1 to 2:1, and further more preferably 20:3 to 10:3. Thisarrangement allows for a significant reduction in concentration of thecryoprotectant as compared with the conventional technique. Accordingly,particularly in a case where the cryopreservation solution is applied toa living body, the cryopreservation solution is safer due to a reductionin the amount of the cryoprotectant entering the living body.

The cryopreservation solution may further contain another component.Examples of the another component can encompass a pH adjusting agent, athickener, and the like. Examples of the pH adjusting agent canencompass sodium hydrogencarbonate, HEPES, a phosphate buffer solution,and the like. Also, in a case where a basic stock solution (BSS) doesnot contain phosphate buffer solution, one added with physiologicalsaline can also be used. Among these components, it is particularlypreferable to use the phosphate buffer solution. It is preferable thatthe pH adjusting agent be used as appropriate in order to adjust the pHof the cryopreservation solution to approximately 6.5 to 9.0, preferablyto 7.0 to 8.5. Note that the phosphate buffer solution in the presentinvention refers to sodium chloride, monosodium phosphate (anhydrous),monopotassium phosphate (anhydrous), disodium phosphate (anhydrous),trisodium phosphate (anhydrous), potassium chloride, potassiumdihydrogen phosphate (anhydrous), and the like, and it is particularlypreferable to use sodium chloride, monosodium phosphate (anhydrous),potassium chloride, or potassium dihydrogen phosphate (anhydrous). Thecontent of the pH adjusting agent in the cryopreservation solution ispreferably 0.01 w/v % to 1.0 w/v %, more preferably 0.05 w/v % to 0.5w/v %.

The cryopreservation solution may contain a thickener. Examples of thethickener can encompass carboxymethyl cellulose (hereinafter, CMC),sodium carboxymethyl cellulose (hereinafter, CMC-Na), organic acidpolymers, propylene glycol alginate, sodium alginate, and the like. Thethickener is preferably CMC or CMC-Na, particularly preferably CMC-Na.Among the organic acid polymers, sodium polyacrylate is preferable.However, since umbilical cord blood or peripheral blood can be preservedwell in the present invention even in a case where the cryopreservationsolution contains no thickener, the cryopreservation solution containsno thickener in a preferable embodiment of the cryopreservation methodin accordance with the present invention. For use as a cell preparationintended for intravenous administration to a human, the cryopreservationsolution desirably contains no thickener.

The cryopreservation solution may or may not contain an animal-derivednatural component. Examples of the animal-derived natural component canencompass albumin, a serum, a basal medium, and the like. Examples ofthe serum can encompass an adult bovine serum, a calf serum, a new borncalf serum, a fetal bovine serum, and the like. Examples of the basalmedium can encompass RPMI medium, MEM medium, HamF-12 medium, DM-160medium, and the like. The cryopreservation solution preferably containsno animal-derived natural component. A cryopreservation solutioncontaining no animal-derived natural component is free of a problem ofvariation in quality from lot to lot of animal-derived naturalcomponents, and also allows avoiding (i) a risk that a componentcontained in the serum and unnecessary for cell preservation, such asvarious cytokines, growth factors, and hormones, may cause a change inthe properties of cells in umbilical cord blood and (ii) an influence ofa component which is contained in the basal medium and whose origin isunknown. Accordingly, particularly for clinical use, thecryopreservation solution containing no animal-derived natural componentis very useful in that the cryopreservation solution is safelyapplicable to a living body.

The cryopreservation solution preferably contains no dextran. Acryopreservation solution containing no dextran allows reducing a riskof causing anaphylactic shock and therefore is safely applicable to aliving body.

The cryopreservation solution preferably contains no hydroxyethylstarch(HES). A cryopreservation solution containing no hydroxyethylstarchallows reducing a risk of causing anaphylactic shock and therefore issafely applicable to a living body.

The cryopreservation solution is preferably an aqueous solution. Theosmotic pressure of the cryopreservation solution is preferably 1000mOsm or more, more preferably 1000 mOsm to 2700 mOsm so that aperformance of the cryopreservation solution as a preservative solutionis retained.

Note that the cryopreservation solution has a composition which may beany combination of the specific examples of components listed above,provided that the composition allows the cryopreservation solution topreserve cells sufficiently. As for concentration, it is also possibleto select and combine concentrations from the specific examples ofconcentrations above.

In a preferable example, the cryopreservation solution is an aqueoussolution which contains a cryoprotectant and glucose and contains noneof an animal-derived natural component, a thickener, dextran, and HES.In a more preferable example, the cryopreservation solution is anaqueous solution which contains DMSO and glucose and contains none of ananimal-derived natural component, a thickener, and dextran. In a furthermore preferable example, the cryopreservation solution is an aqueoussolution which contains DMSO in an amount of 6.0 w/v % to 20.0 w/v % andglucose in an amount of 1.0 w/v % to 10.0 w/v % and contains none of ananimal-derived natural component, a thickener, dextran, and HES. In astill even more preferable example, the cryopreservation solution is anaqueous solution which contains DMSO in an amount of 10.0 v/v % andglucose in an amount of 3.0 w/v % and contains none of an animal-derivednatural component, a thickener, dextran, and HES. In a particularlypreferable example, the cryopreservation solution is an aqueous solutionwhich contains only DMSO, glucose, and a pH adjusting agent. In one ofthe most preferable examples, the cryopreservation solution is anaqueous solution which contains only DMSO, glucose, and a pH adjustingagent, wherein the DMSO is contained in an amount of 6.0 w/v % to 20.0w/v % and the glucose is contained in an amount of 1.0 w/v % to 10.0 w/v%. In another one of the most preferable examples, the cryopreservationsolution is an aqueous solution which contains only DMSO, glucose, and apH adjusting agent, wherein the DMSO is contained in an amount of 10.0v/v % and the glucose is contained in an amount of 3.0 w/v %.

In a case where umbilical cord blood or peripheral blood for medical useis cryopreserved, the cryopreservation solution is preferablysterilized. This is because the sterilized cryopreservation solution hasless risk of bacterial infection and therefore is applicable more safelyto a living body.

Umbilical cord blood or peripheral blood to be cryopreserved may be onefrom which at least part of red blood cells, preferably all of the redblood cells, has been removed. The removal of the red blood cells allowsthe umbilical cord blood or peripheral blood to have a reduced volumewhen frozen, so that a large number of specimens can be preservedeasily. The removal of the red blood cells can be performed, forexample, by a well-known method.

Further, umbilical cord blood or peripheral blood to be cryopreservedmay be one from which at least part of blood plasma has been removed.The removal of the at least part of the blood plasma allows adjustingthe volume of the umbilical cord blood or peripheral blood to be mixedwith the cryopreservation solution. Further, the removal also allows theumbilical cord blood or peripheral blood to have a reduced volume whenfrozen, so that a large number of specimens can be preserved easily. Theremoval of the blood plasma can be performed, for example, by awell-known method.

The cryopreservation solution is mixed with the umbilical cord blood orperipheral blood in a volume ratio of preferably 1:1 to 3:1, morepreferably 1:1 to 2:1, and in an example, further more preferably 1:1.Mixing the cryopreservation solution with the umbilical cord blood orperipheral blood in any of the ranges of volume ratio above allows amixture obtained to have a better cell viability when thawed. Theconcentration of each component in the cryopreservation solution isadjusted in accordance with the above ratios of mixing. For example, ina case where the cryopreservation solution is mixed with the umbilicalcord blood or peripheral blood in a volume ratio of 1:1, theconcentration of each component in the cryopreservation solution shouldbe double the concentration of the each component in the mixture.

The total amount of the cryopreservation solution and the umbilical cordblood or peripheral blood is not particularly limited, but may be, forexample, 10 mL to 100 mL.

The method of mixing is not particularly limited. The mixing ispreferably performed while stirring, from a viewpoint of achieving morehomogenous mixture. The temperature at which the mixing is performed isnot particularly limited but preferably 10° C. or lower, and it is morepreferable that the mixing be performed while icing. Further, thecryopreservation solution is preferably adjusted in advance to thetemperature above. By performing the mixing at such a temperature, it ispossible to suppress an influence of an increase in temperature of themixture and, accordingly, further maintain the cell viability.

The mixture obtained in the mixing step is then frozen. Prior tofreezing, a sample for examination may be collected and the volume ofthe mixture may be measured. Further, the mixture may be transferred toa container which is cold resistant (e.g., a freezer bag). While theseoperations are carried out, the mixture is preferably cooled to 10° C.or lower from a viewpoint of retaining the cell viability.

The freezing step is preferably performed as immediately as possible(e.g., within 30 minutes, preferably within 10 minutes) after the mixingstep. Although the rate of cooling is not particularly limited, slowcooling is preferable. The rate of cooling may be, for example, 1°C./min to 3° C./min. In a case of slow cooling, for example, a programfreezer or the like may be used. The final temperature at which thefreezing is completed is not particularly limited, but preferably −80°C. or lower, more preferably −150° C. or lower, further more preferably−195.8° C. or lower. As the temperature at which the mixture ispreserved is lowered, the mixture can be preserved for a longer periodand in a better condition. Further, the mixture can be frozen at atemperature of approximately −80° C. and then transferred to anenvironment at −180° C. to −200° C. (e.g., in liquid nitrogen) so as tobe preserved. The mixture is preferably frozen in a cold-resistantcontainer.

Umbilical cord blood or peripheral blood which is cryopreserved by thecryopreservation method in accordance with the present invention hasgood cell viability when thawed (see Examples described later). Fromumbilical cord blood or peripheral blood which is cryopreserved by thecryopreservation method in accordance with the present invention, cellscan be recovered, for example, with a cell viability of 80% or more,preferably 85% or more, after an elapse of 2 to 3 months from thefreezing, relative to a cell viability of 100% in the umbilical cordblood or peripheral blood prior to the freezing. Further, from umbilicalcord blood or peripheral blood which is cryopreserved by thecryopreservation method in accordance with the present invention, cellscan be recovered, for example, with a cell viability of 80% or more,preferably 85% or more, after an elapse of 1 month, 3 months, 6 months,1 year, 3 years, 5 years, 10 years, or a longer period (semipermanently)from the freezing, relative to a cell viability of 100% in the umbilicalcord blood or peripheral blood prior to the freezing. The viability ofCD34-positive cells is particularly good, and can be 90% or more,preferably 95% or more, more preferably 98% or more.

As such, in a case where umbilical cord blood or peripheral blood of aperson is cryopreserved by the cryopreservation method in accordancewith the present invention, the umbilical cord blood or peripheral bloodcan be thawed and used in the future when the person or another personreceives a medical care. Further, even if there are unknown cells thatcannot be separated and collected by current technology, thecryopreservation allows such cells to be effectively used when itbecomes possible to isolate the cells in the future. For example, evenfor a disease which is currently untreatable, cryopreserved umbilicalcord blood or peripheral blood can be used in the future when a methodfor treating the disease is developed. Further, for a congenital geneticdisease and the like, cryopreserved umbilical cord blood or peripheralblood can be used as a source of autologous cells for a gene therapy.

Further, umbilical cord blood or peripheral blood cryopreserved by thecryopreservation method in accordance with the present invention, whenthawed, has a good TNC (total nucleated cells) recovery rate, a goodCD34-positive cell recovery rate, and a good total CFU recovery rate(see the Examples described later). From umbilical cord blood orperipheral blood cryopreserved by the cryopreservation method inaccordance with the present invention, cells can be recovered, forexample, with a TNC (total nucleated cells) recovery rate of 92.5% ormore after an elapse of 2 to 3 months from the freezing, relative to aTNC recovery rate of 100% in the umbilical cord blood or peripheralblood prior to the freezing. From umbilical cord blood or peripheralblood cryopreserved by the cryopreservation method in accordance withthe present invention, cells can be recovered, for example, with aCD34-positive cell recovery rate of 67.1% or more after an elapse of 2to 3 months from the freezing, relative to a CD34-positive cell recoveryrate of 100% in the umbilical cord blood or peripheral blood prior tothe freezing. From umbilical cord blood or peripheral bloodcryopreserved by the cryopreservation method in accordance with thepresent invention, cells can be recovered, for example, with a total CFUrecovery rate of 71.9% or more after an elapse of 2 to 3 months from thefreezing, relative to a total CFU recovery rate of 100% in the umbilicalcord blood or peripheral blood prior to the freezing.

According to the present invention, umbilical cord blood or peripheralblood can be cryopreserved well without use of dextran. This allows theumbilical cord blood or peripheral blood to be safely applied to aliving body when thawed. Further, since the cryopreservation solution issimply mixed with umbilical cord blood or peripheral blood without aneed to add anything separately, the umbilical cord blood or peripheralblood can be cryopreserved quickly after being collected.

Note that the present invention encompasses a mixture (composition) of(i) umbilical cord blood or peripheral blood and (ii) thecryopreservation solution which mixture is obtained in the mixing step.

[Method for Thawing Cryopreserved Umbilical Cord Blood or PeripheralBlood]

A method for thawing umbilical cord blood or peripheral blood which hasbeen cryopreserved by the cryopreservation method in accordance with thepresent invention is not particularly limited. A temperature at whichthe thawing is performed is not particularly limited, but preferably ina rage of 30° C. to 40° C., more preferably in a range of 37° C. to 40°C. The thawing is preferably carried out quickly, and may be carriedout, for example, with use of a constant-temperature water bath which isat 37° C. to 38° C.

The thawed umbilical cord blood or peripheral blood can be used formedical, research, or other purposes. In an example, the thawedumbilical cord blood or peripheral blood may be injected as it is into aliving body together with the cryopreservation solution, or injectedinto a living body after the cryopreservation solution is removed.Alternatively, living cells may be collected from the thawed umbilicalcord blood or peripheral blood. The thawed umbilical cord blood, thethawed peripheral blood, or the collected living cells are injected intoa living body, for example, by a method such as intravascularadministration. As discussed above, the thawed umbilical cord blood orperipheral blood has good cell viability and a good cell recovery rate.

As such, the present invention provides a method for administeringumbilical cord blood or peripheral blood which method includes anadministration step of administering the thawed umbilical cord blood orperipheral blood to a living body. Further, the present inventionprovides a method for collecting living cells from umbilical cord bloodor peripheral blood which method includes a cell collection step ofcollecting living cells from the thawed umbilical cord blood orperipheral blood. Further, the present invention provides a method foradministering umbilical cord blood or peripheral blood which methodincludes an administration step of administering, to a living body,living cells collected from the thawed umbilical cord blood orperipheral blood.

In the cell collection step, a method by which the living cells arecollected is not particularly limited, and for example, a well-knownmethod for collecting living cells from umbilical cord blood orperipheral blood may be used. The living cells to be collected are notparticularly limited, and may be, for example, hematopoietic stem cellsor the like. The living cells to be collected are preferably stem cellsor precursor cells, more preferably hematopoietic stem cells.Alternatively, the living cells to be collected may be unknown cellswhich currently cannot be separated. Note that the living cells to becollected are not limited to original cells contained in umbilical cordblood or peripheral blood, and may be cells which were proliferated by aculturing technique (i.e., copies of original cells). The living cellscollected can be used for medical, research, and other purposes. In acase where living cells collected are used for a medical purpose, theliving cells may be transplanted to the person from whom the livingcells collected are derived or to another person.

Further, the present invention encompasses a thawed mixture(composition) of (i) umbilical cord blood or peripheral blood and (ii)the cryopreservation solution.

[Frozen Umbilical Cord Blood or Peripheral Blood, and Method forProducing Same]

A production method in accordance with the present invention forproducing frozen umbilical cord blood or frozen peripheral bloodincludes: a mixing step of mixing a cryopreservation solution withumbilical cord blood or peripheral blood, the cryopreservation solutioncontaining a cryoprotectant and glucose, the umbilical cord blood orperipheral blood containing living cells; and a freezing step offreezing a mixture obtained in the mixing step.

Descriptions for the mixing step and the freezing step are identical tothose given in [Method for cryopreserving umbilical cord blood orperipheral blood].

Frozen umbilical cord blood or peripheral blood which is produced by theproduction method in accordance with the present invention has good cellviability when thawed (see the Examples described later). From frozenumbilical cord blood or peripheral blood in accordance with the presentinvention, cells can be collected, for example, with a cell viability of80% or more after an elapse of 2 to 3 months from the freezing, relativeto a cell viability of 100% in the umbilical cord blood or peripheralblood prior to the freezing. Further, from umbilical cord blood orperipheral blood which is cryopreserved by the cryopreservation methodin accordance with the present invention, cells can be collected, forexample, with a cell viability of 80% or more after an elapse of 1month, 3 months, 6 months, 1 year, 3 years, 5 years, 10 years, or alonger period (semipermanently) from the freezing, relative to a cellviability of 100% in the umbilical cord blood or peripheral blood priorto the freezing.

From the frozen umbilical cord blood or frozen peripheral blood inaccordance with the present invention, living cells may be collected bythe method of collection described above. The living cells collected canbe used for medical, research, and other purposes. The living cells maybe, for example, hematopoietic stem cells or the like. Further, thefrozen umbilical cord blood or frozen peripheral blood in accordancewith the present invention, when thawed, may be injected as it is into aliving body or be injected into a living body after the cryopreservationsolution is removed.

[Cryopreservation Solution for Cryopreserving Umbilical Cord Blood orPeripheral Blood]

A cryopreservation solution in accordance with the present invention forcryopreserving umbilical cord blood or peripheral blood contains acryoprotectant and glucose. Descriptions for the cryopreservationsolution are basically identical to those given in [Method ofcryopreserving umbilical cord blood or peripheral blood].

In an example, the cryoprotectant is contained in the cryopreservationsolution in an amount of preferably 6.0 w/v % to 20.0 w/v %, morepreferably 10.0 w/v % to 16.0 w/v % or 10.0 v/v % to 16.0 v/v %, andfrom a viewpoint of cell recovery rate and reduction in the amount ofDMSO, more preferably 10.0 v/v %.

In an example, the glucose is contained in the cryopreservation solutionin an amount of preferably 0.5 w/v % to 10.0 w/v %, more preferably 1.0w/v % to 10.0 w/v %, further more preferably 2.0 w/v % to 5.0 w/v %, andparticularly preferably 3.0 w/v % to 4.0 w/v %.

[Kit for Cryopreservation]

A kit in accordance with the present invention for cryopreservingumbilical cord blood or peripheral blood includes: the cryopreservationsolution described above for cryopreserving umbilical cord blood orperipheral blood; and a container which is cold resistant and capable ofcontaining (i) the cryopreservation solution and (ii) umbilical cordblood or peripheral blood.

The kit in accordance with the present invention is suitably applicableto [Method for cryopreserving umbilical cord blood or peripheral blood]and [Frozen umbilical cord blood or peripheral blood, and method forproducing same] which are described above.

The container is not particularly limited, provided that the containeris capable of containing the cryopreservation solution and umbilicalcord blood or peripheral blood and is cold resistant. The size of thecontainer is not particularly limited, and can be selected asappropriate in accordance with the amount of the umbilical cord blood orperipheral blood to be contained in the container. The size of thecontainer may be, for example, such that the container has a volume of10 mL to 100 mL, preferably 20 mL to 30 mL. The degree of coldresistance is not particularly limited, provided that the container cantolerate a temperature at which the umbilical cord blood or peripheralblood is preserved. For example, the container is preferably made of amaterial capable of tolerating a temperature of −80° C., more preferablya material capable of tolerating a temperature of −200° C. Examples ofsuch a container encompass a container made of a synthetic resin such aspolyethylene, polypropylene, polystyrene, polyethylene terephthalate,polycarbonate, fluorinated ethylene propylene, and the like. Thecontainer is preferably sealable, and the inside of the container ispreferably sterilized.

The kit in accordance with the present invention may further include aninstruction manual for the kit. The instruction manual for the kit hasrecorded therein the content of the cryopreservation method inaccordance with the present invention described above in the section[Method for cryopreserving umbilical cord blood or peripheral blood]and/or the content of the production method in accordance with thepresent invention described above in the section [Frozen umbilical cordblood or peripheral blood, and method for producing same]. Further, theinstruction manual for the kit may have recorded therein an instructionto use the cryopreservation solution so as to achieve a predeterminedfinal concentration.

[Conclusion]

As described above, a cryopreservation method in accordance with thepresent invention is a cryopreservation method for cryopreservingumbilical cord blood or peripheral blood, including: a mixing step ofmixing a cryopreservation solution with umbilical cord blood orperipheral blood, the cryopreservation solution containing acryoprotectant and glucose; and a freezing step of freezing a mixtureobtained in the mixing step.

The cryopreservation method in accordance with the present invention ispreferably arranged such that the cryopreservation solution contains nothickener.

The cryopreservation method in accordance with the present invention ispreferably arranged such that the cryopreservation solution contains noanimal-derived natural component.

The cryopreservation method in accordance with the present invention ispreferably arranged such that the cryoprotectant is dimethyl sulfoxide.

The cryopreservation method in accordance with the present invention ispreferably arranged such that the mixture contains the cryoprotectant inan amount of 3.0 w/v % to 10.0 w/v % and the glucose in an amount of0.25 w/v % to 5.0 w/v %.

The cryopreservation method in accordance with the present invention ispreferably arranged such that in the mixing step, the cryopreservationsolution is mixed with the umbilical cord blood or the peripheral bloodin a ratio of 1:1 to 3:1.

The cryopreservation method in accordance with the present invention ispreferably arranged such that the umbilical cord blood or the peripheralblood is one from which at least part of red blood cells has beenremoved.

The cryopreservation method in accordance with the present invention ispreferably arranged such that the umbilical cord blood or the peripheralblood is derived from a human.

A production method in accordance with the present invention forproducing frozen umbilical cord blood or frozen peripheral blood is aproduction method for producing frozen umbilical cord blood or frozenperipheral blood, including: a mixing step of mixing a cryopreservationsolution with umbilical cord blood or peripheral blood, thecryopreservation solution containing a cryoprotectant and glucose, theumbilical cord blood or peripheral blood containing living cells; and afreezing step of freezing a mixture obtained in the mixing step.

The present invention also provides frozen umbilical cord blood orfrozen peripheral blood, produced by the production method above.

A cryopreservation solution in accordance with the present invention forcryopreserving umbilical cord blood or peripheral blood contains acryoprotectant and glucose.

A kit in accordance with the present invention is a kit forcryopreserving umbilical cord blood or peripheral blood, including: thecryopreservation solution above for cryopreserving umbilical cord bloodor peripheral blood; and a container which is cold resistant and capableof containing (i) the cryopreservation solution and (ii) umbilical cordblood or peripheral blood.

The embodiments of the present invention will be described in furtherdetail via the following Examples. Needless to say, the presentinvention is not limited to the Examples, and details of the presentinvention can be realized in various manners. Further, the presentinvention is not limited to the description of the embodiments above,but may be altered in various ways within the scope of the appendedClaims. An embodiment based on a proper combination of technical meansdisclosed in different embodiments is encompassed in the technical scopeof the present invention. Furthermore, all the documents describedherein are incorporated herein by reference in their entirety.

EXAMPLES Example 1

[Preparation of Cryopreservation Solutions]

Cryopreservation solution A (Example):

As a cryopreservation solution A, a sterilized aqueous solutioncontaining 10.0 v/v % (11.0 w/v %) of DMSO, 3.0 w/v % of glucose, and asuitable amount of a pH adjusting agent was prepared.

Cryopreservation solution B (Comparative Example):

As a cryopreservation solution B, a sterilized aqueous solutioncontaining 50.0% v/v (55 w/v %) of DMSO and 5.0 w/v % of dextran (Dex40)was prepared.

[Treatment of Umbilical Cord Blood]

(1. Addition of HES)

⅕ of the amount of a raw material umbilical cord blood was calculated asthe volume of hydroxyethylstarch (HES). Then, inside a safety cabinet,an operation adaptor was inserted into a bag for the raw materialumbilical cord blood. The required amount of HES was added using asyringe. The raw material umbilical cord blood and the HES weresufficiently mixed.

(2. Separation of Cells)

A tube of the bag for the raw material umbilical cord blood was clamped,and then connected with a separation bag for cells with use of a sterileconnecting device (TSCD). The bag for the raw material umbilical cordblood was set on a separation stand and left to stand until there was aclear boundary between red blood cells and blood plasma (forapproximately 60 to 90 minutes). When the boundary became clear, thetube was unclamped and the blood plasma and a white blood cell layer(buffy coat) were transferred to the separation bag for cells. At thistime, up to 3 g of a red blood cell layer was allowed to enter theseparation bag for cells. The tube was clamped, and then the bag for theraw material umbilical cord blood and the separation bag for cells weresealed with use of a tube sealer and detached from each other.

(3. Removal of Blood Plasma)

A tip of a tube of a separation bag for blood plasma removal was clampedover a package. Then, the package was opened, and the tube was sealed ata position closer to the bag than the clamped portion (the sterility ofthe bag was maintained until TSCD connection). With confirmation of theseparation bag for cells, the separation bag for blood plasma removalwas marked or labeled with an umbilical cord blood control number, andit was confirmed that the separation bag for cells and the separationbag for blood plasma removal matched in umbilical cord blood controlnumber. A tube of the separation bag for cells was clamped, and thenconnected with the separation bag for blood plasma removal with use of asterile connecting device (TSCD). The separation bag for cells and theseparation bag for blood plasma removal thus connected with each otherwere inserted upright in a bucket of a high capacity centrifuge.Centrifugation was conducted with a centrifugation condition set to400×G and 10 minutes. Upon completion of the centrifugation, theseparation bag for cells was set on a separation stand. The clamp wasremoved, and blood plasma was transferred into the separation bag forblood plasma removal. In doing so, the amount of the blood plasma wasadjusted so that concentrated umbilical cord blood in the separation bagfor cells had a volume of approximately 13.0 mL in a case of mixing theconcentrated umbilical cord blood with the cryopreservation solution A,or a volume of 23.4 mL in a case of mixing the concentrated umbilicalcord blood with the cryopreservation solution B. Tubing between theseparation bag for cells and the separation bag for blood plasma removalwas sealed with use of a tube sealer, and the separation bag for cellsand the separation bag for blood plasma removal were detached from eachother. The weight of the separation bag for cells was measured andrecorded.

(4. Addition of Cryopreservation Solution)

The separation bag for cells was set on an agitator so as to be cooledby a refrigerating agent. The injection rate (flow rate) of a syringepump for the cryopreservation solution A was set to 120 mL/hr, and thesetting was confirmed (level 4). The injection rate (flow rate) of asyringe pump for the cryopreservation solution B was set to 20 mL/hr,and the setting was confirmed. A required amount of the cryopreservationsolution A or the cryopreservation solution B which had been cooled inadvance was dispensed into the syringe. The syringe into which thecryopreservation solution A or the cryopreservation solution B was thusdispensed was fitted with a winged intravenous needle and attached tothe syringe pump. The winged intravenous needle was inserted into anoperation adaptor, and the cryopreservation solution was injected whilebeing cooled and stirred. At this time, the amounts of thecryopreservation solution A and the cryopreservation solution B injectedwere 13.0 mL and 4.4 mL, respectively. As a result, the cryopreservationsolution A when frozen had a final concentration of 5.0 v/v % of DMSOand 1.5 w/v % of glucose, and the cryopreservation solution B whenfrozen had a final concentration of 8.0 v/v % of DMSO and 0.8 w/v % ofdextran (Dex40).

(5. Dispensing of Specimen for Cell Count Measurement)

With confirmation of the umbilical cord blood control number on theseparation bag for cells, a tube for specimen was labeled. An operationadaptor was inserted into the separation bag for cells, the cells insidethe separation bag for cells were suspended uniformly, and 0.5 mL of theumbilical cord blood, which was unfrozen, was dispensed into anexamination tube. Further, 0.5 mL of the unfrozen umbilical cord bloodwas dispensed as a specimen for a sterility test.

(6. Transfer to Freezer Bag)

A plastic needle of a freezer bag was inserted into a port of theseparation bag for cells, and the entire unfrozen umbilical cord bloodwas transferred into the freezer bag. The tube of the freezer bag wasfilled with the unfrozen umbilical cord blood, so that a segment wasproduced. The final weight of the freezer bag was measured, and thenumber of an electronic scale number used and the final weight wererecorded. The unfrozen umbilical cord blood was cooled with arefrigerating agent until a start of a freezing treatment. Note that thefreezing was started within 30 minutes from the completion of theaddition of the cryopreservation solution A.

(7. Freezing)

It was confirmed that the freezer bag, a canister, the segment, and anoperation recording sheet match in umbilical cord blood control number,and the freezer bag was stored in the canister. With use of a programfreezer, the freezing treatment was conducted at a cooling rate of 1°C./min to 3° C./min. At each freezing step, a change in temperature whenfreezing occurred was measured and recorded. Upon completion of thefreezing treatment, the freezer bag was immediately transferred into agas phase of a liquid nitrogen tank.

[Thawing of Cryopreserved Umbilical Cord Blood]

The freezer bag containing umbilical cord blood which had beencryopreserved for 2 to 3 months was put in a constant-temperature waterbath at 35° C. to 37° C. so as to thaw the umbilical cord blood. Theumbilical cord blood thus thawed was iced until a start of each test.

[Examinations Related to Hematopoietic Cells]

(1. Examination of Nucleated Cells)

Examination of WBC (white blood cells) and NRBC (nucleated red bloodcells) was conducted with respect to unfrozen umbilical cord blood andthawed umbilical cord blood. The examination apparatus used was anautomated blood cell counter Sysmex XE-2100, which was based on a flowcytometry technique using a semiconductor laser. The reagents used(Sysmex Corporation) are shown in Table 1.

TABLE 1 Reagent name Dose/specimen Cellpack (II) EPK approx. 30 mL SEsheath (II) ESE approx. 2.1 mL Stromatolyser FB (II) FBT approx. 1.8 mLStromatolyser 4DL FFD approx. 1.8 mL Stromatolyser 4DS FFS approx. 18 μLStromatolyser NR hemolytic agent SNR approx. 1.8 mL Stromatolyser NR dyeSNR approx. 18 μL Sulfolyser SLS approx. 0.5 mL Stromatolyser IM SIMapprox. 3.1 mL RET-search (II) diluent RED approx. 1.8 mL RET-search(II) dye RED approx. 18 μL

The specimen was diluted as appropriate using Cellpack, and was stirredsufficiently. The measurement was conducted in a manual mode. Theexamination data was used to calculate a nucleated cell countconcentration [(WBC+NRBC)×dilution rate]. The nucleated cell countconcentration was used to calculate a TNC (total nucleated cells)recovery rate.

(2. Examination of CD34-Positive Cell Count and Cell Viability)

Examination of CD34-positive cell count and cell viability was conductedwith respect to unfrozen umbilical cord blood and thawed umbilical cordblood. The examination apparatus used was a flow cytometer (hereinafter,FCM) Cytomics FC500 (manufactured by Beckman Coulter, Inc.), which wasbased on a flow cytometry technique using a semiconductor laser.Further, as analysis software, stemCXP software (manufactured by BeckmanCoulter, Inc.) was used. The measurement was conducted with use of a kitfor measuring CD34-positive cell count (StemKit, Beckman Coulter, Inc.,A15573). Other reagents etc. used are shown in Table 2.

TABLE 2 Reagent name IsoFlow sheath fluid Beckman 8599600 Clenz (devicecleaning fluid) Beckman 8546930 Flow check Beckman 6605359

The amount and specimen number of each specimen were confirmed, and thespecimen was diluted with 2% FBS PBS (−). Three tubes (manufactured byBeckman Coulter, Inc., A26428) for the specimen were prepared and markedwith the specimen number and SEQ numbers (1 to 3). To each of the tubes1 and 2, 20 μL of 7-AAD and 20 μL of CD45-FITC/CD34-PE were added. Tothe tube 3, 20 μL of 7-AAD and 20 μL of CD45-FITC were added. To each ofthe tubes 1 to 3, 100 μL of the specimen was added and mixedsufficiently. A mixture thus obtained was left to react in a dark placeat room temperature for 20 minutes. A hemolytic agent (NH₄Cl LysingSolution) was diluted 10-fold with purified water, and added in anamount of 2 mL to each tube (the hemolytic agent was prepared in situ)so as to be mixed sufficiently. A mixture thus obtained was left toreact in a dark place at room temperature for 10 minutes.

The CXP program was activated, and setting of a measurement panel wasconducted. The specimen number etc. were inputted, and the measurementwas started. Upon completion of the measurement, data were confirmedand, if necessary, Gate was corrected by performing a Listmode playback.

On the basis of the analytical data, the concentration and cell count ofCD34-positive cells were calculated. In the case of the thawed umbilicalcord blood, cell viability was also calculated.

(3. Examination of Colony Forming Cell (CFU) Count)

Examination of CFU-GM count and total CFU count was conducted withrespect to the unfrozen umbilical cord blood and the thawed umbilicalcord blood. The reagents (STEMCELL Technologies) are shown in Table 3.

TABLE 3 Reagent name Dose/specimen Medium for CFU: approx. 4 mLMethoCult H4034 Optimum (st04044V) For dilution of specimen: suitableamount 2% FBS-containing IMDM For wetting petri dish: approx. 6 mLSterilized distilled water

MethoCult H4034 Optimum (st04044V) which had been cryopreserved (−15° C.to −25° C.) (approximately 4 mL) was taken out of a freezer, and thawedat or below room temperature. The specimen was diluted with a 2%FBS-containing IMDM medium so as to achieve a nucleated cellconcentration of approximately 400 cells/μL, and was stirredsufficiently. To 4 mL of the thawed MethoCult H4034 Optimum (st04044V),400 μL of the specimen with the nucleated cell concentration thusadjusted was added and stirred intensively with a mixer. A mixture thusobtained was left to stand for approximately 5 minutes until large airbubbles began to rise.

With use of a syringe and needle for methylcellulose, 1.1 mL of thespecimen mixed with the medium was dispensed into each of three 35 mmpetri dishes. The three 35 mm petri dishes were stored in a 90 mm petridish, together with a 35 mm petri dish for distilled water, which 35 mmpetri dish contained approximately 3 mL of sterilized distilled water.The 90 mm petri dish was put in a CO₂ incubator, and cells were culturedunder the conditions of 37° C., 5% CO₂, and 100% humidity for 12 to 15days.

After the culture, a CFU count was counted under an inverted microscope(TS100 (manufactured by Nikon) or IX71-PH (manufactured by Olympus)). Onthe basis of the counted values, a CFU-GM count and a total CFU countwere calculated. With use of the values thus obtained, a CFU-GM recoveryrate and a total CFU recovery rate were calculated.

(4. Results)

Results of the examinations related to hematopoietic cells arecollectively shown in Table 4 (unit: %).

TABLE 4 Cryopreservation Cryopreservation solution A solution B (n = 11)(n = 11) TNC recovery rate 101.9 ± 6.9  101.0 ± 5.4  CD34-positive cellrecovery 95.0 ± 14.8 80.0 ± 8.8  rate

Total CFU recovery 95.7 ± 16.8 85.7 ± 12.5 CFU-GM recovery rate 92.3 ±21.5 92.7 ± 17.9 Living cell viability 86.8 ± 2.9  88.6 ± 7.9 (fluorescent microscope) AO/EB Living cell viability 74.0 ± 6.1  71.4 ±9.8  (Flow) CD45 site Living cell viability 99.4 ± 0.7  99.1 ± 0.8 (Flow) CD34 site

Example 2

[Preparation of Cryopreservation Solutions]

Cryopreservation solution C (Example):

As a cryopreservation solution C, a sterilized aqueous solutioncontaining 16.0 v/v % (17.6 w/v %) of DMSO, 3.0 w/v % of glucose, and asuitable amount of a pH adjusting agent was prepared.

Cryopreservation solution B (Comparative Example):

As a cryopreservation solution B, a sterilized aqueous solutioncontaining 50.0% v/v (55 w/v %) of DMSO and 5.0 w/v % of dextran (Dex40)was prepared.

[Treatment of Umbilical Cord Blood]

The treatment of umbilical cord blood was conducted in accordance with asimilar procedure as in Example 1. Note that the cryopreservationsolution C when frozen had a final concentration of 8.0 v/v % of DMSOand 1.5 w/v % of glucose, and the cryopreservation solution B whenfrozen had a final concentration of 8.0 v/v % of DMSO and 0.8 w/v % ofdextran (Dex40).

[Thawing of Cryopreserved Umbilical Cord Blood]

The freezer bag containing umbilical cord blood which had beencryopreserved for 2 months was put in a constant-temperature water bathat 35° C. to 37° C. so as to thaw the umbilical cord blood. Theumbilical cord blood thus thawed was iced until a start of each test.

[Examinations Related to Hematopoietic Cells]

Examinations related to hematopoietic cells were conducted in a similarmanner to Example 1.

(Results)

Results of the examinations related to hematopoietic cells arecollectively shown in Table 5 (unit: %). Table 5 also shows resultswhich were recalculated from the results in Table 4 of Example 1 inconsideration of a difference between the cryopreservation solution Aand the cryopreservation solution B in timing of collecting umbilicalcord blood. Note that the results of the cryopreservation solution C inTable 5 were calculated in consideration of a timing of collectingumbilical cord blood. As for the cryopreservation solution B, resultsobtained in Example 1 and results obtained in Example 2 are merged inTable 5, since the same evaluation method was employed between Examples1 and 2.

TABLE 5 Cryopreservation Cryopreservation Cryopreservation solution Asolution B solution C (n = 11) (n = 21) (n = 15) TNC recovery 101.2 ±7.1  100.0 ± 7.1  99.0 ± 4.1  rate CD34-positive 96.5 ± 13.1 83.0 ± 9.186.9 ± 9.2  cell recovery rate

Total CFU 98.3 ± 19.1  95.6 ± 19.5 94.3 ± 18.0 recovery CFU-GM 92.5 ±23.7  99.7 ± 23.9 97.0 ± 22.3 recovery rate Living cell 86.8 ± 2.9  85.7± 7.6 85.2 ± 2.8  viability (fluorescent microscope) AO/EB Living cell74.0 ± 6.1  75.2 ± 9.7 84.2 ± 2.6 viability (Flow) CD45 site

Living cell 89.4 ± 0.7  98.1 ± 1.7 98.8 ± 1.1  viability (Flow) CD34site

Example 3

With use of a same specimen, a comparative experiment ofcryopreservation of umbilical cord blood was conducted.

[Preparation of Cryopreservation Solutions]

Cryopreservation solution A (Example):

As a cryopreservation solution A, a sterilized aqueous solutioncontaining 10.0 v/v % (11.0 w/v %) of DMSO, 3.0 w/v % of glucose, and asuitable amount of a pH adjusting agent was prepared.

Cryopreservation solution C (Example):

As a cryopreservation solution C, a sterilized aqueous solutioncontaining 16.0 v/v % (17.6 w/v %) of DMSO, 3.0 w/v % of glucose, and asuitable amount of a pH adjusting agent was prepared.

[Treatment of Umbilical Cord Blood]

Red blood cells were removed in accordance with a similar procedure asin Example 1, and centrifugal concentration was conducted until theumbilical cord blood had a volume of 26.5 mL. From the umbilical cordblood thus concentrated, 0.5 mL was collected as a sample of unfrozenumbilical cord blood, and the remaining portion was divided in half(13.0 mL). Each half was mixed with 13.0 mL of the cryopreservationsolution A or the cryopreservation solution C in accordance with asimilar procedure as in Example 1, and then cryopreserved in accordancewith a similar procedure as in Example 1. The cryopreservation solutionA when frozen had a final concentration of 5.0 v/v % of DMSO and 1.5 w/v% of glucose, and the cryopreservation solution C when frozen had afinal concentration of 8.0 v/v % of DMSO and 1.5 w/v % of glucose.

[Thawing of Cryopreserved Umbilical Cord Blood]

The freezer bag containing umbilical cord blood which had beencryopreserved for 2 months was put in a constant-temperature water bathat 35° C. to 37° C. so as to thaw the umbilical cord blood. Theumbilical cord blood thus thawed was iced until a start of each test.

[Examinations Related to Hematopoietic Cells]

Examinations related to hematopoietic cells were conducted in a similarmanner to Example 1.

(Results)

Results of the examinations related to hematopoietic cells arecollectively shown in Table 6 (unit: %).

TABLE 6 Cryopreservation Cryopreservation solution A solution C (n = 10)(n = 10) TNC recovery rate 100.9 ± 8.3  97.6 ± 5.9 

CD34-positive cell recovery  92.9 ± 11.1 83.2 ± 15.4 rate

Total CFU recovery  87.5 ± 25.2 82.0 ± 20.4 CFU-GM recovery rate  87.9 ±27.1 76.8 ± 25.0 Living cell viability  88.2 ± 4.9  86.2 ± 6.5 (fluorescent microscope) AO/EB Living cell viability  75.9 ± 5.8  82.8 ±5.2  (Flow) CD45 site

Living cell viability  98.3 ± 3.7  98.5 ± 1.0  (Flow) CD34 site

As compared with the cryopreservation solution C, the cryopreservationsolution A had higher cell recovery rates. Also from a viewpoint ofreduction in the amount of transfused DMSO, the cryopreservationsolution A is considered preferable to the cryopreservation solution C.

INDUSTRIAL APPLICABILITY

The present invention is applicable to cryopreservation of umbilicalcord blood and peripheral blood for medical, research, and otherpurposes.

1. A cryopreservation method for cryopreserving umbilical cord blood orperipheral blood, comprising: a mixing step of mixing a cryopreservationsolution with umbilical cord blood or peripheral blood, thecryopreservation solution containing a cryoprotectant and glucose; and afreezing step of freezing a mixture obtained in the mixing step.
 2. Thecryopreservation method as set forth in claim 1, wherein thecryopreservation solution contains no thickener.
 3. The cryopreservationmethod as set forth in claim 1, wherein the cryopreservation solutioncontains no animal-derived natural component.
 4. The cryopreservationmethod as set forth in claim 1, wherein the cryoprotectant is dimethylsulfoxide.
 5. The cryopreservation method as set forth in claim 1,wherein the mixture contains the cryoprotectant in an amount of 3.0 w/v% to 10.0 w/v % and the glucose in an amount of 0.25 w/v % to 5.0 w/v %.6. The cryopreservation method as set forth in claim 1, wherein in themixing step, the cryopreservation solution is mixed with the umbilicalcord blood or the peripheral blood in a volume ratio of 1:1 to 3:1. 7.The cryopreservation method as set forth in claim 1, wherein theumbilical cord blood or the peripheral blood is one from which at leastpart of red blood cells has been removed.
 8. The cryopreservation methodas set forth in claim 1, wherein the umbilical cord blood or theperipheral blood is derived from a human.
 9. A production method forproducing frozen umbilical cord blood or frozen peripheral blood,comprising: a mixing step of mixing a cryopreservation solution withumbilical cord blood or peripheral blood, the cryopreservation solutioncontaining a cryoprotectant and glucose, the umbilical cord blood orperipheral blood containing living cells; and a freezing step offreezing a mixture obtained in the mixing step.
 10. Frozen umbilicalcord blood or frozen peripheral blood, produced by a method recited inclaim
 9. 11. A cryopreservation solution for cryopreserving umbilicalcord blood or peripheral blood, comprising a cryoprotectant and glucose.12. A kit for cryopreserving umbilical cord blood or peripheral blood,comprising: a cryopreservation solution recited in claim 11; and acontainer which is cold resistant and capable of containing (i) thecryopreservation solution and (ii) umbilical cord blood or peripheralblood.